Quercetin protects RAW264.7 macrophages from glucosamine-induced apoptosis and lipid accumulation via the endoplasmic reticulum stress pathway.

It is increasingly recognized that macrophages are a key cell in the development of atherosclerosis. Glucosamine, the product of the hexosamine biosynthetic pathway in diabetes mellitus, can disturb lipid metabolism, induce apoptosis and accelerate atherosclerosis via endoplasmic reticulum (ER) stress in various types of cells. Previous studies have indicated that quercetin possesses antidiabetic, anti­oxidative, anti­inflammatory and anti­apoptotic activities as a flavonoid. Studies have also demonstrated its novel pharmacological properties for inhibiting ER stress. The present study focussed on the effects of quercetin on cell injury and ER stress in glucosamine­induced macrophages. RAW264.7 macrophages were cultured with 15 mM glucosamine, following which the levels of apoptosis, intracellular total and free cholesterol, and apoptosis­ and ER stress­associated proteins were measured in the macrophages treated with or without quercetin. Additionally, the ratio of cholestryl ester/total cholesterol was calculated to observe the formation of foam cells. The results demonstrated that apoptosis and abnormal lipid accumulation in the RAW264.7 cells, which was induced by glucosamine, were significantly reversed by quercetin. In addition, quercetin treatment suppressed the increase of C/EBP homologous protein, and inhibited the activation of JNK and caspase­12, which was induced by glucosamine. Quercetin also increased the expression level of full length activating transcriptional factor 6 and decreased the expression of glucose regulated protein 78. Of note, the beneficial effects of quercetin on the glucosamine­induced RAW264.7 cells were reversed by treatment with tunicamycin. These findings suggest that quercetin may have properties to prevent glucosamine­induced apoptosis and lipid accumulation via the ER stress pathway in RAW264.7 macrophages.

Effects of grape seed proanthocyanidin extract on renal injury in type 2 diabetic rats.

Grape seed proanthocyanidin extract (GSPE) is known to be an effective natural polyphenol capable of removing free radicals in vivo. It has been reported that GSPE has biological functions including antioxidant, anti-cancer, anti-hyperglycemic, anti-radiation, and prevention and treatment of cardiovascular diseases. This study aims to investigate the effects of GSPE on renal injury in type 2 diabetic rats induced with low-dose streptozotocin and a high-carbohydrate/high-fat diet. Rats (n=12 per group) were administered GSPE at either a low (125 mg/kg · bw), medium (250 mg/kg · bw) or high (500 mg/kg · bw) dose, while control rats and diabetes mellitus group rats received no specific treatment. After 16 weeks, GSPE slightly increased body weight and decreased food consumption, water intake and urine volume in rats. Diabetic rats treated with GSPE demonstrated decreased fasting blood glucose, serum insulin, HbA1c and systolic blood pressure (P<0.05). GSPE significantly improved renal function parameters, reduced the expression of tissue inhibitor of metalloproteinase-1 and also increased the activity of matrix metalloproteinase-9. Moreover, GSPE (particularly at a dose of 500 mg/kg · bw) increased the activity of antioxidant enzymes and reduced the levels of c-reactive proteins (P<0.01) in serum and the expression of tumor necrosis factor-α, monocyte chemoattractant protein-1 and intercellular adhesion molecule-1 (P<0.05) in the kidney. These findings suggest that GSPE ameliorates renal injury in type 2 diabetic rats through its antioxidative activity and anti-inflammatory effects.

Comparative studies of oral administration of marine collagen peptides from Chum Salmon (Oncorhynchus keta) pre- and post-acute ethanol intoxication in female Sprague-Dawley rats.

The present study aimed to evaluate the effect of an oral administration of marine collagen peptides (MCPs) pre- and post-acute ethanol intoxication in female Sprague-Dawley (SD) rats. MCPs were orally administered to rats at doses of 0 g per kg bw, 2.25 g per kg bw, 4.5 g per kg bw and 9.0 g per kg bw, prior to or after the oral administration of ethanol. Thirty minutes after ethanol treatment, the effect of MCPs on motor incoordination and hypnosis induced by ethanol were investigated using a screen test, fixed speed rotarod test (5 g per kg bw ethanol) and loss of righting reflex (7 g per kg bw ethanol). In addition, the blood ethanol concentrations at 30, 60, 90, and 120 minutes after ethanol administration (5 g per kg bw ethanol) were measured. The results of the screen test and fixed speed rotarod test suggested that treatment with MCPs at 4.5 g per kg bw and 9.0 g per kg bw prior to ethanol could attenuate ethanol-induced loss of motor coordination. Moreover, MCP administered both pre- and post-ethanol treatment had significant potency to alleviate the acute ethanol induced hypnotic states in the loss of righting reflex test. At 30, 60, 90 and 120 minutes after ethanol ingestion at 5 g per kg bw, the blood ethanol concentration (BEC) of control rats significantly increased compared with that in the 4.5 g per kg bw and 9.0 g per kg bw MCP pre-treated groups. However, post-treatment with MCPs did not exert a significant inhibitory effect on the BEC of the post-treated groups until 120 minutes after ethanol administration. Therefore, the anti-inebriation effect of MCPs was verified in SD rats with the possible mechanisms related to inhibiting ethanol absorption and facilitating ethanol metabolism. Moreover, the efficiency was better when MCPs were administered prior to ethanol.

Grape seed proanthocyanidin extracts ameliorate podocyte injury by activating peroxisome proliferator-activated receptor-γ coactivator 1α in low-dose streptozotocin-and high-carbohydrate/high-fat diet-induced diabetic rats.

Podocytes are part of the glomerular filtration membrane in kidney and serve to prevent the filtration of protein from the blood. Several evidences suggest that mitochondrial dysfunction plays a critical role in the pathogenesis of diabetic nephropathy and it is an early event in podocyte injury. Mitochondrial dysfunction promotes oxidative stress that can favor the development of podocyte injury. Peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α) was considered to be a major regulator of metabolic homeostasis and mitochondrial function. Some studies indicated that polyphenols may improve mitochondrial dysfunction, maintain the podocyte integrity and have therapeutic effects on glomerular diseases by promoting PGC-1α expression. Our study investigated whether grape seed proanthocyanidin extracts (GSPE), a strong antioxidant, ameliorate podocyte injury by activating PGC-1α in low-dose streptozotocin-and high-carbohydrate/high-fat diet-induced diabetic rats. After 16 weeks of GSPE treatment, GSPE slightly increased the body weight and decreased plasma glucose, food intake, water intake and urine volume in diabetic rats. Further, GSPE significantly decreased 24 h albumin levels and increased the expression of nephrin and podocalyxin. The antioxidant levels were improved and the cellular damage of kidney in diabetic rats was also relieved effectively after the treatment. Moreover, GSPE increased the mRNA expression of mitochondrial biogenesis factors and mitochondrial DNA content. Finally, GSPE activated the expression of PGC-1α, silent mating type information regulation 2 homolog 1 (SIRT1) and AMP-activated protein kinase (AMPK). These results suggest that GSPE ameliorate podocyte injury in diabetic nephropathy by the activation of AMPK-SIRT1-PGC-1α signalling, which appears to inhibit oxidative stress and mitochondrial dysfunction in the kidney.

Functional and morphological effects of grape seed proanthocyanidins on peripheral neuropathy in rats with type 2 diabetes mellitus.

Grape seed proanthocyanidins (GSPs) possess a broad spectrum of pharmacological and therapeutic properties. The aim of this study was to examine the effect of GSPs on functional and morphological abnormalities in the peripheral nerves of rats with type 2 diabetes mellitus. Diabetic rats were induced by two injections of 25 mg streptozotocin/kg body weight and 8 weeks of a high-carbohydrate/high-fat diet. GSPs were then administrated to the rats for 16 weeks. Thermal and mechanical sensitivity thresholds and nerve conductive velocity were measured to evaluate peripheral nerve function. Light microscopy was used with special stains to observe the morphological changes in the central and peripheral nervous systems. Calcium (Ca(2+)) homeostasis and ATPase activities in the sciatic nerves were also determined. In diabetic rats receiving GSP treatment (especially at the 500 mg/kg dose), the abnormal peripheral nerve function and impaired nervous tissues (L4 to L5 spinal cord segments, L5 dorsal root ganglion, and sciatic nerves) were improved to a significant extent. Moreover, 500 mg/kg GSP treatment significantly reduced the concentration of free Ca(2+) and elevated Ca(2+)-ATPase activity in sciatic nerves. These results suggest that GSPs may prevent early functional and morphological abnormalities in the peripheral nerves of rats with type 2 diabetes mellitus.

Quercetin but not quercitrin ameliorates tumor necrosis factor-alpha-induced insulin resistance in C2C12 skeletal muscle cells.

Skeletal muscle is a major site for glucose metabolism and its injury by cytokines can induce insulin resistance leading to type 2 diabetes. It has been suggested that quercetin may act as an anti-diabetic agent, however, the effects of quercetin on insulin resistance in skeletal muscle remain unknown. We aimed to investigate the role of quercetin and its glycoside, quercitrin in tumor necrosis factor-alpha (TNF-α) induced C2C12 skeletal muscle cell impairment. Quercetin, but not quercitrin moderately attenuated the effects of TNF-α and enhanced the basal and insulin stimulated uptake of glucose in a dose-dependent manner via the activation of the protein kinase B (Akt) and AMP-activated protein kinase (AMPK) pathways. Furthermore, the underlying mechanism also involved the suppression of nuclear factor-κB (NF-κB) signaling and the nitric oxide (NO)/inducible nitric oxide synthase (iNOS) system, downstream of AMPK transduction. In summary, quercetin exhibited its effect of improving glucose uptake and insulin sensitivity in skeletal muscle cells via the two independent signaling pathways of Akt and AMPK, and can be developed as a potential anti-diabetic agent.

Quercetin and quercitrin protect against cytokine­induced injuries in RINm5F ß-cells via the mitochondrial pathway and NF-κB signaling.

Quercetin, existing mostly in its glycoside form quercitrin, is the most widely distributed flavonoid in nature. It possesses various potential effects as an antioxidant, anti-inflammatory for cell damage of ß-cells, however, studies on this topic are limited and controversial. In order to examine the effects of quercetin on type I diabetes mellitus, we investigated the role of quercetin/quercitrin in cytokine-induced ß-cell injuries in RINm5F rat insulinoma cells. Cell viability, glucose-stimulated insulin secretion (GSIS), intracellular reactive oxygen species (ROS), nitric oxide (NO) and inflammation or apoptosis-associated protein expression were measured with or without quercetin/quercitrin treatment. We also compared the differences between the aglycone and the glycoside forms of quercetin, with the aim to shed some light on their structures and transportation into cells. The results showed that quercetin/quercitrin protected against cytokine-induced cell death, improved GSIS, and inhibited ROS as well as NO accumulation. These effects were associated with reduced expression of inducible nitric oxide synthases (iNOS) and inhibited translocation of nuclear factor-κB (NF-κB). Also, quercetin/quercitrin suppressed cytochrome c release from mitochondria and the following alteration of downstream proteins, suggesting that mitochondrial apoptosis was attenuated by quercetin treatment. In summary, quercetin and quercitrin are potential candidates to prevent ß-cell death via the mitochondrial pathway and NF-κB signaling, and quercetin may be more efficacious than quercitrin as an anti-diabetic agent.

Myricetin protects against cytokine-induced cell death in RIN-m5f ß cells.

Cytokine-induced cell death is recognized as a major cause of progressive ß-cell loss. Tumor necrosis factor α (TNF-α), interleukin 1ß (IL-1ß), and interferon γ (IFN-γ) in combination trigger a series of events that lead to ß-cell death. In the past few decades, the use of myricetin as an anti-inflammatory and cytoprotective agent has gained much attention. The present study focused on the protective roles of myricetin against cytokine-induced cell death in insulin-secreting RIN-m5f ß cells. The results showed that myricetin (especially at concentrations of 10 µM and 20 µM) increased cell viability and decreased cell apoptosis induced by the cytokine mixture of TNF-α (10 ng/mL), IL-1ß (5 ng/mL), and IFN-γ (1000 IU/mL) for 3 days. Moreover, the cytokines increased the total and p65 subunit levels of nuclear factor κB, decreased inhibitor κB α levels, stimulated the accumulation of nitric oxide, increased cytochrome c release from mitochondria, and induced reactive oxygen species generation; myricetin (especially at the concentration of 20 µM) abolished all of these parameters. These results suggest that myricetin might have therapeutic value for preventing ß-cell death.

Effect of polysaccharide from cultured Cordyceps sinensis on immune function and anti-oxidation activity of mice exposed to 60Co.

AIM OF THE STUDY: The present study was designed to investigate the molecular weight (MW), chemical composition and effect of polysaccharide (CS-PS), from the fruiting bodies of cultured Cordyceps sinensis, on immune function and anti-oxidation activity of BALB/c mice exposed to (60)Co. MATERIALS AND METHODS: The MW of CS-PS was determined by gel-filtration. The chemical composition of CS-PS was tested by using gas chromatography-mass spectrophotometer (GC-MS). Mice were administered CS-PS with doses of 50, 100 or 200mg/kg body weight, then exposed to (60)Co. The normal control group and irradiated control group were also used. Four days later, lymphocyte proliferation, activity of macrophage phagocytosis, delayed type hypersensitivity (DTH), concentration of malondialdehyde (MDA), total-superoxide dismutase (SOD) enzyme activity, and cytokine expression in serum from the mice were tested. RESULTS: The average molecular weight of CP-PS was 12 kD. The polysaccharide was composed of mannose, rhamnose, arabinose, xylose, glucose and galactose. Lymphocyte proliferation, the activity of macrophage phagocytosis, DTH and total-SOD enzyme activity in the CS-PS groups were significantly enhanced compared to the irradiated control group. Lipid peroxidation level was significantly reduced in the CS-PS groups compared to the irradiated control group. Levels of cytokine IL-4, IL-5 and IL-17 are also affected in the CS-PS groups compared to the irradiated control group. CONCLUSIONS: CS-PS, a heteropolysaccharide, enhances immunity activity in mice treated by ionizing radiation, through reducing oxidative injury and modulating the secretion of cytokine IL-4, IL-5 and IL-17.

Epigallocatechin-3-gallate protects pro-inflammatory cytokine induced injuries in insulin-producing cells through the mitochondrial pathway.

Pro-inflammatory cytokine-mediated pancreatic ß-cell dysfunction is a key pathological event in type 1 diabetes mellitus. There are few studies about the protection of epigallocatechin-3-gallate (EGCG) against pro-inflammatory cytokine-induced ß-cell apoptosis. To examine the direct effects of EGCG on ß-cells, insulin-producing RINm5F cells were exposed to a combination of recombinant interleukin-1 beta (IL-1ß), tumor necrosis factor alpha (TNF-α), and interferon gamma (IFN-γ), with or without EGCG pretreatment for 24h. Cell death was monitored by the MTT assay. Glucose-stimulated insulin release was measured using radio immunoassay. Intracellular reactive oxygen species was examined with dichlorofluorescein (DCF) fluorescence by flow cytometry. To evaluate RINm5F cells mitochondrial function, change in mitochondrial membrane potential, intracellular ATP levels, and nitric oxide was assessed. The expression of cytochrome c, Bax, Bcl-2, and iNOS proteins was measured by western blotting. In the present study, EGCG pretreatment protected against cytokines inducing cell death and restored glucose stimulated-insulin secretion in RINm5F cells. EGCG reduced the cytokine-induced generation of reactive oxygen species, the loss of mitochondrial membrane potential (Δψm), the release of cytochrome c from the mitochondria, and translocation of Bax protein to the mitochondria from the cytosol. EGCG pretreatment prevented cytokine-induced iNOS overexpression and NO generation. In summary, pro-inflammatory cytokines lead to a reduction of glucose-induced insulin secretion, mitochondrial activity and viability in RINm5F cells. The pro-inflammatory cytokine-induced effects can be prevented by EGCG pretreatment via the mitochondrial pathway.

Oral administration of skin gelatin isolated from Chum salmon (Oncorhynchus keta) enhances wound healing in diabetic rats.

Care for diabetic wounds remains a significant clinical problem. The present study was aimed at investigating the effect of skin gelatin from Chum Salmon on defective wound repair in the skin of diabetic rats. Full-thickness excisional skin wounds were made in 48 rats, of which 32 were diabetes. The diabetic rats were orally treated daily for 14 days with skin gelatin from Chum Salmon (2 g/kg) or its vehicle. Sixteen non-diabetic control rats received the same amount of water as vehicle-treated non-diabetic rats. Rats were killed to assess the rate of wound closure, microvessel density (MVD), vascular endothelial growth factor (VEGF), hydroxyproline (HP) contents in wound tissues and nitrate in plasma and wound tissue at 7 and 14 days after wounding. Skin gelatin-treated diabetic rats showed a better wound closure, increased MVD, VEGF, hydroxyproline and NO contents and a reduced extent of inflammatory response. All parameters were significant (P < 0.05) in comparison to vehicle-treated diabetic group. In light of our finding that skin gelatin of Chum Salmon promotes skin wound repair in diabetic rats, we propose that oral administration of Chum Salmon skin gelatin might be a beneficial method for treating wound disorders associated with diabetes.

Oral administration of marine collagen peptides from Chum Salmon skin enhances cutaneous wound healing and angiogenesis in rats.

BACKGROUND: A wound is a clinical entity which often poses problems in clinical practice. The present study was aimed to investigate the wound healing potential of administering marine collagen peptides (MCP) from Chum Salmon skin by using two wound models (incision and excision) in rats. RESULTS: Ninety-six animals were equally divided into the two wound models and then within each model animals were randomly divided into two groups: vehicle-treated group and 2 g kg(-1) MCP-treated group. Wound closure and tensile strength were calculated. Collagen deposition was assessed by Masson staining and hydroxyproline measurement. Angiogenesis was assessed by immunohistological methods. MCP-treated rats showed faster wound closure and improved tissue regeneration at the wound site, which was supported by histopathological parameters pertaining to wound healing. MCP treatment improved angiogenesis and helped form thicker and better organised collagen fibre deposition compared to vehicle-treated group. CONCLUSION: The results show the efficacy of oral MCP treatment on wound healing in animals.

Prevention of retinoic acid-induced early craniofacial abnormalities by vitamin B12 in mice.

OBJECTIVE: The purpose of the present study was to identify the potential effect of prenatal vitamin B12 administration on retinoic acid (RA)-induced early craniofacial abnormalities in mice and to investigate the possible mechanisms by which vitamin B12 reduces malformations. DESIGN: In our study, whole embryo culture was used to explore the effect of vitamin B12 on mouse embryos during the critical period of organogenesis. All embryos were exposed to 0.4 µM RA and different concentrations of vitamin B12 and scored for their growth in the branchial region at the end of a 48-hour culture period. The endothelin-1 (ET-1)/dHAND protein expression levels in the first branchial arch were investigated using an immunohistochemical method. RESULTS: In the whole embryo culture, 100 and 10 µM vitamin B12 dose-dependently prevented branchial region malformations and decreased craniofacial defects by 90.5% and 77.3%, respectively. ET-1 and dHAND protein levels were significantly increased in vitamin B12-supplemented embryos compared to the RA-exposed group in embryonic branchial region. CONCLUSIONS: These results suggest that vitamin B12 may prevent RA-induced craniofacial abnormalities via prevention of an RA-induced decrease of ET-1 and dHAND protein levels in the branchial region during the organogenic period. This study may shed new light on preventing craniofacial abnormalities.

[Establishment of insulin-resistant muscle cell model induced by tumor necrosis factor-alpha].

OBJECTIVE: To establish a convenient and reliable insulin-resistant cell model induced by TNF-alpha for studying the mechanisms of insulin-resistance and screening medical drugs or functional foods assisted in improving insulin-resistance. METHODS: L6 cells in TNF-alpha group, insulin group and TNF-alpha plus insulin group were improving incubated in culture medium containing 10 ng/ml TNF-alpha, 100 nmol/L insulin and both 10 ng/ml TNF-alpha and 100 nmol/L insulin respectively for 24 hours. The glucose remained in culture medium was measured by the method of glucose oxidizes peroxides (GOD-POD). The glucose uptake of L6 cells was measured by [3H]-2-deoxyglucose. RESULTS: With no insulin stimulation, the glucose remained in culture medium in TNF-alpha group was equal to that in the control group (P > 0.05). With insulin stimulation, the glucose remained in culture medium in insulin group was much lower than that in TNF-alpha group (P < 0.05). CONCLUSION: The insulin resistance in L6 cells could be induced by high concentration of TNF-alpha. The IR model of skeletal muscle cell could be widely used for studying the mechanism of insulin resistance and for screening medical drugs and functional food assisted in reducing blood sugar.

[Effect of acetyl-L-carnitine on the insulin resistance of L6 cells induced by tumor necrosis factor-alpha].

OBJECTIVE: To investigate the effects of acetyl-L-carnitine (ALC) on the insulin resistance of rat L6 cells induced by tumor necrosis factor-alpha (TNF-alpha). METHODS: Well-differentiated rat L6 muscle cells were divided into 6 groups, including control group, insulin (100 nmol/L) group, insulin (100 nmol/L) + TNF-alpha (10 ng/ml) group, and three insulin + TNF-alpha + ALC (0.1, 0.3 and 0.6 mmol/L respectively) groups. The glucose remained in culture medium was measured by the method of glucose oxidizes peroxides (GOD-POD). The glucose uptake of L6 cells was tested by [3H]-2-deoxyglucose. The expression of insulin receptor substrate-1 (IRS-1) was examined by western blotting. RESULTS: The glucose content remained in culture medium was increased by TNF-alpha and the glucose uptake of cells was inhibited by TNF-alpha. The inhibition of TNF-alpha could be improved by ALC and in a dose-response relation. Compared with the insulin group, the expression of Ser307 phosphorylation of IRS-1 was lower in TNF-alpha group, and the expression of Ser307 phosphorylation of IRS-1 was decreased by ALC. CONCLUSION: The insulin resistance in L6 cells induced by TNF-alpha might be attenuated by ALC via decreasing the expression of Ser307 phosphorylation of IRS-1.